Functional analysis of two promoters for the human mitochondrial glycerol phosphate dehydrogenase gene

Qiuming Gong, Laura J. Brown, Michael J. MacDonald

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Mitochondrial glycerol phosphate dehydrogenase (mGPD) is abundant in the normal pancreatic insulin cell, but its level is lowered 50% by diabetes. To evaluate mGPD expression, we cloned and characterized the 5'-flanking region of the human mGPD gene. The gene has two alternative first exons and two promoters. The downstream promoter (B) is 10 times more active than the upstream promoter (A) in insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher activity in INS-1 than in non-β cells. Deletion and mutation analysis suggested that a NRF-2 binding site at -94 to -101 and an E2F binding site at -208 to -215 are important regulatory cis elements in promoter B. Gel mobility shift assays indicated that the -94 to -101 region binds the NRF-2 protein. When INS-1 cells were maintained in the presence of high glucose (25 mM) for 7 days, mGPD was the only 1 of 6 enzyme activities lowered (53%). mGPD promoter B activity was reduced by 60% in INS-1 cells by the high glucose, but in HepG2 cells and HeLa cells, promoter B activity was unchanged or slightly increased. Deletion analysis indicated the glucose responsiveness was distributed across the region from -340 to -260 in promoter B. The results indicate that mGPD gene transcription in the beta cell is regulated differently from other cells and that decreased mGPD promoter B transcription is at least in part the cause of the decreased beta cell mGPD levels in diabetes.

Original languageEnglish (US)
Pages (from-to)38012-38021
Number of pages10
JournalJournal of Biological Chemistry
Volume275
Issue number48
DOIs
StatePublished - Dec 1 2000
Externally publishedYes

Fingerprint

glycerol dehydrogenase
Functional analysis
Glycerol
Oxidoreductases
Genes
Phosphates
Transcription
Medical problems
HeLa Cells
Glucose
Binding Sites
Insulin
Sequence Deletion
5' Flanking Region
Hep G2 Cells
Insulin-Secreting Cells
Enzyme activity
Electrophoretic Mobility Shift Assay
Exons
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional analysis of two promoters for the human mitochondrial glycerol phosphate dehydrogenase gene. / Gong, Qiuming; Brown, Laura J.; MacDonald, Michael J.

In: Journal of Biological Chemistry, Vol. 275, No. 48, 01.12.2000, p. 38012-38021.

Research output: Contribution to journalArticle

@article{186b1be4f0014864aea8d12903c7c9ec,
title = "Functional analysis of two promoters for the human mitochondrial glycerol phosphate dehydrogenase gene",
abstract = "Mitochondrial glycerol phosphate dehydrogenase (mGPD) is abundant in the normal pancreatic insulin cell, but its level is lowered 50{\%} by diabetes. To evaluate mGPD expression, we cloned and characterized the 5'-flanking region of the human mGPD gene. The gene has two alternative first exons and two promoters. The downstream promoter (B) is 10 times more active than the upstream promoter (A) in insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher activity in INS-1 than in non-β cells. Deletion and mutation analysis suggested that a NRF-2 binding site at -94 to -101 and an E2F binding site at -208 to -215 are important regulatory cis elements in promoter B. Gel mobility shift assays indicated that the -94 to -101 region binds the NRF-2 protein. When INS-1 cells were maintained in the presence of high glucose (25 mM) for 7 days, mGPD was the only 1 of 6 enzyme activities lowered (53{\%}). mGPD promoter B activity was reduced by 60{\%} in INS-1 cells by the high glucose, but in HepG2 cells and HeLa cells, promoter B activity was unchanged or slightly increased. Deletion analysis indicated the glucose responsiveness was distributed across the region from -340 to -260 in promoter B. The results indicate that mGPD gene transcription in the beta cell is regulated differently from other cells and that decreased mGPD promoter B transcription is at least in part the cause of the decreased beta cell mGPD levels in diabetes.",
author = "Qiuming Gong and Brown, {Laura J.} and MacDonald, {Michael J.}",
year = "2000",
month = "12",
day = "1",
doi = "10.1074/jbc.M004078200",
language = "English (US)",
volume = "275",
pages = "38012--38021",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "48",

}

TY - JOUR

T1 - Functional analysis of two promoters for the human mitochondrial glycerol phosphate dehydrogenase gene

AU - Gong, Qiuming

AU - Brown, Laura J.

AU - MacDonald, Michael J.

PY - 2000/12/1

Y1 - 2000/12/1

N2 - Mitochondrial glycerol phosphate dehydrogenase (mGPD) is abundant in the normal pancreatic insulin cell, but its level is lowered 50% by diabetes. To evaluate mGPD expression, we cloned and characterized the 5'-flanking region of the human mGPD gene. The gene has two alternative first exons and two promoters. The downstream promoter (B) is 10 times more active than the upstream promoter (A) in insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher activity in INS-1 than in non-β cells. Deletion and mutation analysis suggested that a NRF-2 binding site at -94 to -101 and an E2F binding site at -208 to -215 are important regulatory cis elements in promoter B. Gel mobility shift assays indicated that the -94 to -101 region binds the NRF-2 protein. When INS-1 cells were maintained in the presence of high glucose (25 mM) for 7 days, mGPD was the only 1 of 6 enzyme activities lowered (53%). mGPD promoter B activity was reduced by 60% in INS-1 cells by the high glucose, but in HepG2 cells and HeLa cells, promoter B activity was unchanged or slightly increased. Deletion analysis indicated the glucose responsiveness was distributed across the region from -340 to -260 in promoter B. The results indicate that mGPD gene transcription in the beta cell is regulated differently from other cells and that decreased mGPD promoter B transcription is at least in part the cause of the decreased beta cell mGPD levels in diabetes.

AB - Mitochondrial glycerol phosphate dehydrogenase (mGPD) is abundant in the normal pancreatic insulin cell, but its level is lowered 50% by diabetes. To evaluate mGPD expression, we cloned and characterized the 5'-flanking region of the human mGPD gene. The gene has two alternative first exons and two promoters. The downstream promoter (B) is 10 times more active than the upstream promoter (A) in insulin-secreting cells (INS-1) and HeLa cells. Promoter B has higher activity in INS-1 than in non-β cells. Deletion and mutation analysis suggested that a NRF-2 binding site at -94 to -101 and an E2F binding site at -208 to -215 are important regulatory cis elements in promoter B. Gel mobility shift assays indicated that the -94 to -101 region binds the NRF-2 protein. When INS-1 cells were maintained in the presence of high glucose (25 mM) for 7 days, mGPD was the only 1 of 6 enzyme activities lowered (53%). mGPD promoter B activity was reduced by 60% in INS-1 cells by the high glucose, but in HepG2 cells and HeLa cells, promoter B activity was unchanged or slightly increased. Deletion analysis indicated the glucose responsiveness was distributed across the region from -340 to -260 in promoter B. The results indicate that mGPD gene transcription in the beta cell is regulated differently from other cells and that decreased mGPD promoter B transcription is at least in part the cause of the decreased beta cell mGPD levels in diabetes.

UR - http://www.scopus.com/inward/record.url?scp=0034528195&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034528195&partnerID=8YFLogxK

U2 - 10.1074/jbc.M004078200

DO - 10.1074/jbc.M004078200

M3 - Article

C2 - 10954707

AN - SCOPUS:0034528195

VL - 275

SP - 38012

EP - 38021

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 48

ER -